This invention relates to a multi-dimensional optical design. More particularly the invention relates to an optical design of a multi-dimensional system which can simultaneously detect five or more distinct properties of particles or cells when the design is applied to a flow cytometric analyzer.
Particle analysis, known generally as flow cytometry, consists of passing particles one at a time through a sensing region of a flowcell, and detecting the properties or characteristics, of each particle. These specific properties, which are sometimes referred to as dimensions, are usually combinations of multi-angle light scatter and multi-color fluorescence.
Flow cytometry has become a particularly important method for analyzing blood cells in the hematology laboratory where patient test load is an important metric. This is because the method is rapid, enabling as many as five to ten thousand cells per second to be analyzed, and because it is much more statistically accurate than the manual microscope inspection method. It is important, however, to the hematology laboratory, that the entire process, both sample preparation and analysis, be automated.
A large number of products exist today which feature such multi-dimensional capability, but only a few automate the entire process. Two of the most well known such products in which the entire process of blood cell analysis, or differentiation is fully automated are the Cell-Dyn.RTM. series 3000 and 3500 analyzers manufactured by Abbott Diagnostics. Each of these instruments measures simultaneously four dimensions which include three angles of laser light scatter, and a fourth dimension which is depolarized light scatter.
A number of products exist which measure several simultaneous dimensions of fluorescence and scatter in which only the analysis is automated. One of the most well known of these is the Becton Dickinson FACScan.RTM. flow cytometer. This instrument is capable of simultaneously detecting one dimension of forward scatter, one dimension of side scatter, and three colors of fluorescence.
However, in none of these multi-dimensional products which combine several colors of fluorescence and light scatter, is the entire process automated. Part of the reason for this is the complexity of building a system which is stable enough to maintain proper alignment for many simultaneous dimensions while at the same time, assuring the measurement integrity of each cell or particle in the sample stream for all dimensions.
Among the prior art contributions, is the Auer et al. U.S. Pat. No. 4,038,556 which describes a two-dimensional system with a flowcell, a laser light source, and two simultaneous optical paths, a side angle collection system for measuring cell fluorescence, and a forward angle system for measuring light scatter. The patent teaches that by placing the forward angle detector in the back focus of a light collecting lens, an important and practical simplification of system alignment results; the precise relationship of the forward angle optical system, with respect to the remaining elements of the system, is greatly relaxed. Although the side angle beam focus, the laser beam focus, and the stream focus must be established to be mutually collinear in the Auer et al. teachings, it is not required for the forward angle path. This is due to design of the forward path system which transforms the two dimensional distribution of intensity vs angular distribution in the flowcell space to intensity vs spatial distribution at the detector.
Hirako, in U.S. Pat. No. 4,953,979, describes a side angle collection system for flow cytometry which has the PMT front surface conjugate with the condenser exit pupil while the flow stream (containing the particles or cells) is conjugate with an external aperture located between the condenser and the PMT. The external aperture, which limits unwanted background light, is located at the front focus of a second lens, which functions to image the condenser exit pupil on the PMT. The patent teaches that as the stream position, or cell position within the stream varies, the effect on cell coefficient of variant ("C.V.") of detector sensitivity variations are eliminated.
Hirako, ignores the C.V. effect of stream or cell position variations within the flowcell upon the angular integrity of the scattered light with respect to the laser beam.
It is one object of this invention to maintain the angular integrity of the scattered light with respect to the laser beam in both the forward and side angle light paths.
It is another object of this invention to improve the stability of, and at the same time simplify, the alignment and tracking requirements of a multi-dimensional flow cytometer.
It is yet another object of this invention to combine this design approach with a multi-element array detector and a simple laser beam translating mechanism, to assure beam to stream tracking simplification, while at the same time assuring the measurement integrity of each particle or cell, independent of cell location in the stream, or the precise stream location within the flowcell.
It is another object of this invention to maximize these advantages in at least two separate light paths simultaneously.
These and other advantages will become more apparent in the following detailed description.